这项工作得到了国家卫生研究院（R01 DK077195至R.M.A、R01 DK104641至R.M.A和D.R.B）的赠款支持。我们感谢斯图亚特·奥金博士在血液学/肿瘤学系、波士顿儿童医院、儿科系、哈佛医学院和达纳-法伯癌症研究所提供的宝贵意见。我们还感谢凯尔·科斯塔、玛丽·塔利恩蒂和哈比巴拉·肖贾伊萨迪博士（儿科部杨石博士实验室、新生儿医学部、儿科部、新生儿医学部、波士顿儿童医院、哈佛医学院）在技术援助和有益讨论方面给予的支持。

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没有宣布任何利益冲突。

此处描述的鼠脊髓损伤模型提供了一种可重复的方法，由于膀胱收缩和外部尿道括约肌松弛之间失去协调，可产生功能性膀胱出口阻塞。这反过来又唤起了膀胱壁的深刻改造，早在受伤后2周，其特点是扩大尿道和光滑的肌肉隔间。在啮齿动物中实施SCI模型的关键步骤包括:（i） 在脊柱休克期间严格注意手动膀胱表达，在受伤后10-u201214天;（二） 营养丰富，以尽量减少体重减轻:（三） 缓解尿液烫伤的可能性，特别是用于超越反射失效返回的实验。该模型的局限性包括:在瞬时血尿期间，小鼠可能从血栓中排出尿道，另外，在手术后逆行射精后，来自精液库古鲁姆的雄性小鼠中。
此处描述的组织分离方法说明了考虑实验侮辱引起的组织结构变化的重要性，在这种情况下，SCI 之后的重大组织重塑可能会影响下游分析。随着单细胞分析的增加，必须确保在基因表达中观察到的差异不仅仅是分离引起的扰动的结果，而是真正代表与疾病模型相关的潜在生物变化。使用公开的表达数据使我们能够修改消化缓冲器的配方，以确保细胞外矩阵的有效消化，同时最大限度地提高可行性。在未来的应用中可以考虑的其他修改包括添加actinomycin D，以阻止对分离协议15敏感的即时早期基因的转录。
在分离组织或转移已经处于悬浮状态的细胞时，管道技术至关重要。为了减少剪切力对细胞的物理损害，在细胞重新悬念期间轻轻和缓慢地使用移液器非常重要。一般建议使用宽孔移液器提示。如果使用标准提示，则轻轻使用移液器细胞悬架，以避免剪切力，否则会损害细胞，这一点尤为重要。然而，使用细胞过滤器是不可避免的，但是，细胞浓度可以下降20%或更多，伴随着100μL或以上的体积损失。我们建议在紧张后确定细胞浓度，以确保精确的细胞计数。
在流细胞学中，FMO 控制提供背景的测量，因为信号从重叠的发射峰值出血。它们不是非特异性抗体结合的量度，也不是当抗体包含在该通道中时可能存在的背景染色。要考虑非特异性抗体结合，必须包括适当的同型控制:对于背景染色，需要包括负控制。综合起来，这些控制确保准确测量细胞群。

正常尿囊功能的扰动会导致许多人生活质量下降。为了更好地了解损伤或疾病如何破坏膀胱的正常功能，重要的是要探索膀胱内细胞的正常生物状态，以及它们在实验扰动下是如何变化的。然而，迄今为止，尿囊内的特定细胞群以及它们如何随损伤而变化，其特征尚未完全确定。
单细胞剖析方法，如流细胞切除术或单细胞RNA测序（scRNA-seq），有可能揭示膀胱内的特定细胞类型。然而，要使这些方法成为信息性组织，必须以不影响收获组织的生存能力、基因表达和代表性细胞群百分比的方式进行消化。采用酶分聚的方案可以通过不分青红皂白的蛋白酶活性1影响表面标记表达，从而通过流细胞学影响细胞识别，而分离过程本身可导致立即早期基因的诱导，正如范登布林克及其同事最近描述的那样。作者表明，虽然受分离影响的亚人口很少，但由于即时早期基因的表达水平较高，它可能会在批量表达研究中触发强烈的污染信号。此外，分离协议的持续时间影响了某些亚种群所特有的基因的体积表达水平。因此，在不考虑分离协议影响的情况下生成的单个细胞数据集可能会产生分离方法产生的基因表达变化，而不是基础生物学。这些观察表明，发表的单细胞转录经济学数据应谨慎解释，结果应通过独立方法进行验证。
虽然，苛刻和冗长的分离方法可能会改变细胞2中的基因表达:有效分离细胞对于准确表示存在的细胞类型至关重要。由于膀胱是一个复杂的器官，由多种细胞类型组成，因此一些群体，如尿道细胞或频闪细胞，可能相对代表不足，而其他细胞类型，如成纤维细胞存在于细胞外基质内，可能难以分离。分离变得更加具有挑战性，如果膀胱经历了重大的改造和纤维化，如在脊髓损伤3，4或膀胱出口障碍5，6观察到。
在这里，我们描述了脊髓损伤小鼠膀胱下游单细胞分析的优化组织分离方法。使用流细胞学，我们比较了四种酶消化方案，它们能够产生单个细胞悬浮，支持细胞存活率，并保持细胞群的正确比例。基于这一分析，我们得出结论，尽量减少细胞死亡、细胞聚集物、非细胞核酸和下游分析的潜在抑制剂对于实现高质量的数据至关重要。

<table><tbody><tr><td>2.5 X Magnifying Loupes</td><td></td><td></td><td></td></tr><tr><td>7-0 Vicryl suture, 6.5mm needle 3/8 circle</td><td>ETHICON</td><td>J546</td><td></td></tr><tr><td>70 &amp;mu;m Cell Strainer</td><td>Thermofisher</td><td>22363548</td><td></td></tr><tr><td>Accutase in BPBS, 0.5mM EDTA</td><td>Millipore</td><td>SCR005</td><td></td></tr><tr><td>Aerosol Filter Wide Orifice Pipettor Tips (1000 &amp;micro;L)</td><td>VWR</td><td>89049-168</td><td></td></tr><tr><td>Aerosol Filter Wide Orifice Pipettor Tips (1000 &amp;micro;L)</td><td>VWR</td><td>89049-168</td><td></td></tr><tr><td>APC anti-mouse CD326 (Ep-CAM), rat monoclonal, IgG2a, &amp;kappa;, affinity purified</td><td>BioLegend</td><td>118213</td><td></td></tr><tr><td>BB515 Rat Anti-Mouse CD45, rat monoclonal, IgG2b, &amp;kappa;, Clone 30-F11</td><td>BD Biosciences</td><td>564590</td><td></td></tr><tr><td>BONN Micro Dissecting Forceps, Straight, 1x2 teeth, 3.75&quot; length, 0.3mm tip width, 0.12mm teeth</td><td>ROBOZ Surgical Instrument Company, Inc.</td><td>RS-5172</td><td>ROBOZ Surgical Instrument Company, Inc., Gaithersburg MD</td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>A9647-100G</td><td></td></tr><tr><td>CaCl2</td><td>Sigma</td><td>2115-250ML</td><td></td></tr><tr><td>CASTROVIEJO Micro Suturing Needle Holder, Straight with lock, 5.75&quot; length</td><td>ROBOZ Surgical Instrument Company, Inc.</td><td>RS-6412</td><td>ROBOZ Surgical Instrument Company, Inc., Gaithersburg MD</td></tr><tr><td>Cell Counting Kit, 30 dual-chambered slides, 60 counts, with trypan blue</td><td>Biorad</td><td>1450003</td><td></td></tr><tr><td>Cell Staining Buffer</td><td>BioLegend</td><td>420201</td><td></td></tr><tr><td>Collagenase from Clostridium histolyticum</td><td>Sigma</td><td>C0130-1G</td><td></td></tr><tr><td>Collagenase Type I</td><td>Worthington Biochemical Corporation</td><td>LS004196</td><td></td></tr><tr><td>Collagenase Type III</td><td>Worthington Biochemical Corporation</td><td>LS004182</td><td></td></tr><tr><td>Collagenase, Type 6</td><td>Worthington Biochemical Corporation</td><td>LS005319</td><td></td></tr><tr><td>Dead Cell Apoptosis Kit with Annexin V Alexa Fluor 488 &amp;amp; Propidium Iodide (PI)</td><td>Thermofisher</td><td>V13241</td><td></td></tr><tr><td>Dispase II</td><td>Sigma</td><td>D4693-1G</td><td></td></tr><tr><td>DNase</td><td>Sigma</td><td>DN25-1G</td><td></td></tr><tr><td>Enrofloxacin (Baytril)</td><td>Bayer Health Care LLC,</td><td>NADA # 140-913 Approved by FDA. Lot No.: AH01CGP</td><td>2.27% Injectable Solution</td></tr><tr><td>Falcon 15 ml conical centrifuge tubes</td><td>Fisher Scientific</td><td>352096</td><td></td></tr><tr><td>Falcon 50 ml conical centrifuge tubes</td><td>Fisher Scientific</td><td>352070</td><td></td></tr><tr><td>FITC anti-mouse Ly-6A/E (Sca-1) Antibody, rat monoclonal, IgG2a, &amp;kappa;, affinity purified</td><td>BioLegend</td><td>122505</td><td></td></tr><tr><td>Hyaluronidase from sheep testes, Type II</td><td>Sigma</td><td>H2126</td><td></td></tr><tr><td>MACS SmartStrainers (100 &amp;micro;m)</td><td>Miltenyi Biotec, Inc.</td><td>130-110-917</td><td></td></tr><tr><td>McPHERSON-VANNAS, Micro Dissecting Spring Scissors, Straight, 4&quot; length, 0.15mm tip width</td><td>ROBOZ Surgical Instrument Company, Inc.</td><td>RS-5630</td><td>ROBOZ Surgical Instrument Company, Inc., Gaithersburg MD</td></tr><tr><td>Meloxicam</td><td>Patterson Veterinary</td><td>07-891-7959</td><td></td></tr><tr><td>Papain</td><td>Worthington Biochemical Corporation</td><td>LS003119</td><td></td></tr><tr><td>PE/Cy5 anti-mouse CD19 Antibody, rat monoclonal, IgG2a, &amp;kappa;, affinity purified</td><td>BioLegend</td><td>115509</td><td>Dump Channel</td></tr><tr><td>PE/Cy5 anti-mouse CD3&amp;epsilon; Antibody, Armenian hamster monoclonal, IgG, affinity purified</td><td>BioLegend</td><td>100309</td><td>Dump Channel</td></tr><tr><td>PE/Cy5 anti-mouse CD4 Antibody, rat monoclonal, IgG2b, &amp;kappa;, affinity purified</td><td>BioLegend</td><td>100409</td><td>Dump Channel</td></tr><tr><td>PE/Cy5 anti-mouse CD8a Antibody, rat monoclonal, IgG2a, &amp;kappa;, affinity purified</td><td>BioLegend</td><td>100709</td><td>Dump Channel</td></tr><tr><td>PE/Cy5 anti-mouse NK-1.1 Antibody, mouse monoclonal, IgG2a, &amp;kappa;, affinity purified</td><td>BioLegend</td><td>108715</td><td>Dump Channel</td></tr><tr><td>PE/Cy5 anti-mouse TER-119/Erythroid Cells Antibody, IgG2b, &amp;kappa;, affinity purified</td><td>BioLegend</td><td>116209</td><td>Dump Channel</td></tr><tr><td>Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block), rat monoclonal, IgG2b, &amp;kappa;, Clone 2.4G2</td><td>BD Biosciences</td><td>553141</td><td></td></tr><tr><td>RBC Lysis Buffer (10X)</td><td>BioLegend</td><td>420301</td><td></td></tr><tr><td>Red Blood Cell Lysis Buffer 1x</td><td>Biolegend</td><td>420201</td><td></td></tr><tr><td>Screw-Cap microcentrifuge tubes, 1.5 ml</td><td>VWR</td><td>89004-290</td><td></td></tr><tr><td>TC20 Automated Cell Counter</td><td>Biorad</td><td>1450102</td><td></td></tr><tr><td>Triple antibiotic ointment (neomycin/polymyxin B/ bacitracin)</td><td>Patterson Veterinary</td><td>07-893-7216</td><td>skin protectant</td></tr><tr><td>TrypLE Select Enzyme (10X), no phenol red</td><td>Thermofisher</td><td>A1217701</td><td></td></tr><tr><td>Vetropolycin eye ointment</td><td>Dechra Veterinary Products</td><td>NADA # 065-016. Approved by FDA.</td><td>protect eyes during anesthesia</td></tr></tbody></table>

a single cell dissociation approach for molecular analysis of urinary bladder in the mouse following spinal cord injury

我们描述了脊髓损伤在小鼠的实施，以引起脱毛器括约肌发育不良，功能性膀胱出口阻塞，以及随后的膀胱壁改造。为了促进评估非损伤控制和脊髓损伤小鼠膀胱壁的细胞组成，我们开发了一个优化的分离方案，支持高细胞存活率，并允许通过流细胞检测离散亚人口。
脊髓损伤是由胸脊髓的完全转位造成的。在组织收获时，动物在深度麻醉下注入磷酸盐缓冲盐水，膀胱被收获到Tyrode的缓冲液中。组织在消化缓冲区孵化之前切碎，消化缓冲区根据小鼠膀胱的胶原蛋白含量进行优化，通过对公开的基因表达数据库的询问确定。单细胞悬浮后，通过流细胞学分析材料，以评估细胞存活率、细胞数和特定子群落。我们证明，该方法产生细胞群超过90%的生存能力，并有力地表示细胞的间质和上皮起源。这种方法将能准确分析小鼠膀胱和其他器官中的离散细胞类型。

手术 胸脊髓转选的成功取决于对若干参数的评估，其中最明显的是后肢瘫痪。动物只用前肢移动，拖着后肢。否则，活动水平，包括喂养，梳妆和警觉性通常是正常的。此外，动物失去意志性膀胱控制，导致研究者每12小时需要手动膀胱表达，直到反射无效，受伤后10至14天返回。安乐死后，损伤成功的额外迹象主要与膀胱与体重比的增加有关，表明组织重塑。组理分析显示尿道和光滑的肌肉隔间3内增生。
准备单细胞悬架 利用公开的表达数据，确定细胞外基质蛋白的膀胱组织富集（图2），用于为消化组合的配方提供信息。由于胶原蛋白是膀胱壁11，12的关键组成部分，首先我们试图确定最丰富的胶原蛋白（s）在鼠标膀胱使用RNA分析数据集产生的鼠标ENCODE项目13。我们的分析表明，胶原蛋白1A1、胶原蛋白3A1、胶原蛋白1A2和胶原蛋白6A1是小鼠膀胱内最丰富的胶原蛋白类型（图2A）。我们还使用 Tabula Muris（鼠标（肌肉）的单细胞转录体数据简编）8来确定胶原蛋白 1、3、6 和透明质的 mRNA 表达水平。这些数据允许直接和对组织之间共享的细胞类型的基因表达进行对比。分析表明，这些细胞外基质组件的表达在间质细胞类型中更为普遍，而不是尿素（图2B）。
分离对膀胱中分离细胞生存能力的影响 流细胞学分析表明，采用4种不同方案的酶消化的存活率分别为83%、86%、93%和90%。因此，协议第3节被认为对保持细胞存活性最有价值。我们还观察到，大约4%的细胞是坏死细胞（PI+/附件V-）（图4A）。这些观察强调了消化方案的效率以及随后对细胞生存能力的好处。
脊髓损伤对膀胱中不同细胞群的影响 与对照组相比，我们观察到SCI小鼠膀胱的总细胞数量显著增加。从SCI膀胱获得的点图模式也略有不同，与脊髓损伤引起的器官重塑（图4B:第一列）一致。与对照组相比，SCI动物的膀胱显示CD45阳性细胞显著增加。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61455/61455fig01.jpg"> 图1:代表输液完成与肝脏的浅色。（A） 在输液开始时显示肝脏颜色。（B） 在输液结束时显示减轻的肝脏颜色。（A）中的小鼠在输液前两周进行脊髓转选，导致膀胱肥大，其突出出骨盆不像（B）中没有脊髓损伤的鼠标:在这种情况下，膀胱很小，隐藏在骨盆里。 <a href="https://www.jove.com/files/ftp_upload/61455/61455fig01large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61455/61455fig02.jpg"> 图2:小鼠膀胱细胞外基质（ECM）组件的转录表达。（A） 43种不同胶原蛋白类型的条形图。该表达方式由每千万个映射读数（RPKM）（数据来自生物项目:PRJNA66167）14表示。（B） 在一池男性和女性分离的尿囊样本（男性和女性）中，从基于微流体液滴的3'end计数中获得的细胞类型的小提琴基因表达图谱。使用每百万 ln （CPM+1）8的自然对数，每个单元的计数均进行日志规范化。在进行对数之前添加了 1 个 CPM 的伪计数。<a href="https://www.jove.com/files/ftp_upload/61455/61455fig02large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61455/61455fig03.jpg"> 图3:控制策略和FMO控制，以确定荧光扩散。（A） 细胞种群的选择。（B） 单打的格子策略。（C） 使用PI和附件V抗体对坏死细胞和早晚凋亡细胞进行治疗。（D_2012F）多色流细胞学的示意图点图（例如，与 A、B、C、D 荧光素 + （附件 V 和 PI）结合的抗体）。与未染色控制相比，FMO 控制显示的荧光通过荧光 A 通道扩散到抗体中。橙色虚线表示 FMO 界点边界，而未染色的边界为红色。 <a href="https://www.jove.com/files/ftp_upload/61455/61455fig03large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61455/61455fig04.jpg"> 图4:膀胱中不同细胞类型的流细胞学。（A） 附件 V/PI 双染色流程图。每个协议所使用的酶和化学成分的不同组合在相应的生存图前表示。这些数据表明，通过协议第 3 节获得了最高的可行性。（B） 代表直方图，说明在单个通道中检测到的 Ly-6A/E （Sca-1） 和 CD326 （Ep-CAM） 的强度。（ C） SCI对小鼠膀胱细胞群的影响。上面板显示从对照非手术小鼠获得的三个分离膀胱上的染色结果，下面板显示三个带有SCI的动物染色的结果。第一栏是细胞总数。第二栏显示单项网格选择。第三栏显示了对B细胞、T细胞和NK细胞呈阴性的活细胞的亚群。第四栏显示活细胞的染色对CD45呈阳性。 <a href="https://www.jove.com/files/ftp_upload/61455/61455fig04large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>组件</td>
			<td>金额（500 mL）</td>
			<td>摩尔 浓度</td>
		</tr>
<tr>
<td>Nacl</td>
			<td>4.091 克</td>
			<td>140米</td>
		</tr>
<tr>
<td>氯化钾</td>
			<td>0.186 克</td>
			<td>5米</td>
		</tr>
<tr>
<td>MgCl2
</td>
			<td>0.0476 克</td>
			<td>1米</td>
		</tr>
<tr>
<td>D-葡萄糖</td>
			<td>0.9 克</td>
			<td>10米</td>
		</tr>
<tr>
<td>海佩斯</td>
			<td>1.19 克</td>
			<td>10米</td>
		</tr>
</tbody></table>表1:泰罗德解决方案准备的组件。 指示的组件用于准备 500 mL 泰罗德的解决方案。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>组件</td>
			<td>量</td>
			<td>协议第 1 节</td>
			<td>协议第2节</td>
			<td>协议第3节</td>
			<td>协议第4节</td>
		</tr>
<tr>
<td>Bsa</td>
			<td>5毫克</td>
			<td>是的</td>
			<td>是的</td>
			<td>是的</td>
			<td>是的</td>
		</tr>
<tr>
<td>卡克2
</td>
			<td>0.03 mM</td>
			<td>是的</td>
			<td>是的</td>
			<td>是的</td>
			<td>是的</td>
		</tr>
<tr>
<td>拼贴酶 I 型</td>
			<td>132.5 单位</td>
			<td>是的</td>
			<td>是的</td>
			<td>是的</td>
			<td>是的</td>
		</tr>
<tr>
<td>拼贴酶类型 I</td>
			<td>96.4 单位</td>
			<td>是的</td>
			<td>是的</td>
			<td>是的</td>
			<td>是的</td>
		</tr>
<tr>
<td>拼贴酶类型六</td>
			<td>50 个单位</td>
			<td>-</td>
			<td>-</td>
			<td>是的</td>
			<td>-</td>
		</tr>
<tr>
<td>纳塞</td>
			<td>10 个单位</td>
			<td>是的</td>
			<td>是的</td>
			<td>是的</td>
			<td>-</td>
		</tr>
<tr>
<td>木瓜 蛋白酶</td>
			<td>115 单位</td>
			<td>-</td>
			<td>-</td>
			<td>是的</td>
			<td>是的</td>
		</tr>
<tr>
<td>潘拼贴画</td>
			<td>50 个单位</td>
			<td>-</td>
			<td>-</td>
			<td>是的</td>
			<td>是的</td>
		</tr>
<tr>
<td>透明 质 酸 酶</td>
			<td>10.5 单位</td>
			<td>-</td>
			<td>-</td>
			<td>是的</td>
			<td>是的</td>
		</tr>
<tr>
<td>迪斯帕塞二世</td>
			<td>1.25 单位</td>
			<td>是的</td>
			<td>是的</td>
			<td>-</td>
			<td>-</td>
		</tr>
<tr>
<td>细胞分离溶液</td>
			<td>1 mL</td>
			<td>是的</td>
			<td>-</td>
			<td>是的</td>
			<td>-</td>
		</tr>
<tr>
<td>重组酶</td>
			<td>1 mL</td>
			<td>-</td>
			<td>是的</td>
			<td>-</td>
			<td>是的</td>
		</tr>
</tbody></table>表2:用于准备消化缓冲液的组件。 所示组件用于制备 2.5 mL 消化混合物（1 U 催化 1 μmol 每分钟 37 °C 的基板水解。 有关每个酶单位的定义，请参阅产品数据表）。

Watch this Scientific Journal Video about A Single Cell Dissociation Approach for Molecular Analysis of Urinary Bladder in the Mouse Following Spinal Cord Injury at JoVE.com

A Single Cell Dissociation Approach for Molecular Analysis of Urinary Bladder in the Mouse Following Spinal Cord Injury

此协议的目标是将优化的组织分离协议应用于脊髓损伤的鼠标模型，并验证通过流细胞学进行单细胞分析的方法。

脊髓损伤后小鼠泌尿膀胱分子分析的单细胞分离方法

We describe the implementation of spinal cord injury in mice to elicit detrusor-sphincter dyssynergia, a functional bladder outlet obstruction, and ...

a-single-cell-dissociation-approach-for-molecular-analysis-urinary

Research

JoVE Journal

Biology

5.5K 次观看.  Boston Children's Hospital. 此协议的目标是将优化的组织分离协议应用于脊髓损伤的鼠标模型，并验证通过流细胞学进行单细胞分析的方法。

视频: A Single Cell Dissociation Approach for Molecular Analysis of Urinary Bladder in the Mouse Following Spinal Cord Injury

Mouse Bladder Wall Injection

Mouse bladder wall injection is a useful technique to orthotopically study bladder phenomena, including stem cell, smooth muscle, and cancer biology. Before starting injections, the surgical area must be cleaned with soap and water and antiseptic solution. Surgical equipment must be sterilized before use and between each animal. Each mouse is placed under inhaled isoflurane anesthesia (2-5% for induction, 1-3% for maintenance) and its bladder exposed by making a midline abdominal incision with scissors. If the bladder is full, it is partially decompressed by gentle squeezing between two fingers. The cell suspension of interest is intramurally injected into the wall of the bladder dome using a 29 or 30 gauge needle and 1 cc or smaller syringe. The wound is then closed using wound clips and the mouse allowed to recover on a warming pad. Bladder wall injection is a delicate microsurgical technique that can be mastered with practice.

Mouse bladder wall injection is a useful approach to orthotopically study bladder stem cell and cancer biology. This delicate microsurgical method can be mastered with careful technique and practice.

鼠标膀胱壁注射液

Mouse bladder wall injection is a useful technique to orthotopically study bladder phenomena, including stem cell, smooth muscle, and cancer biology. ...

科研

医学

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher frequency of fractures 1. As these fractures often occur in regions that have lost normal sensory function, the patient is at a greater risk of fracture-dependent pathologies, including death. SCI-dependent loss in both bone mineral density (BMD, grams/cm2) and bone mineral content (BMC, grams) has been attributed to mechanical disuse 2, aberrant neuronal signaling 3 and hormonal changes 4. The use of rodent models of SCI-induced osteoporosis can provide invaluable information regarding the mechanisms underlying the development of osteoporosis following SCI as well as a test environment for the generation of new therapies 5-7 (and reviewed in 8). Mouse models of SCI are of great interest as they permit a reductionist approach to mechanism-based assessment through the use of null and transgenic mice. While such models have provided important data, there is still a need for minimally-invasive, reliable, reproducible, and quantifiable methods in determining the extent of bone loss following SCI, particularly over time and within the same cohort of experimental animals, to improve diagnosis, treatment methods, and/or prevention of SCI-induced osteoporosis.
An ideal method for measuring bone density in rodents would allow multiple, sequential (over time) exposures to low-levels of X-ray radiation. This study describes the use of a new whole-animal scanner, the IVIS Lumina XR (Caliper Instruments) that can be used to provide low-energy (1-3 milligray (mGy)) high-resolution, high-magnification X-ray images of mouse hind limb bones over time following SCI. Significant bone density loss was seen in the tibiae of mice by 10 days post-spinal transection when compared to uninjured, age-matched control (na&iuml;ve) mice (13% decrease, p&lt;0.0005). Loss of bone density in the distal femur was also detectable by day 10 post-SCI, while a loss of density in the proximal femur was not detectable until 40 days post injury (7% decrease, p&lt;0.05). SCI-dependent loss of mouse femur density was confirmed post-mortem through the use of Dual-energy X-ray Absorptiometry (DXA), the current "gold standard" for bone density measurements. We detect a 12% loss of BMC in the femurs of mice at 40 days post-SCI using the IVIS Lumina XR. This compares favorably with a previously reported BMC loss of 13.5% by Picard and colleagues who used DXA analysis on mouse femurs post-mortem 30 days post-SCI 9. Our results suggest that the IVIS Lumina XR provides a novel, high-resolution/high-magnification method for performing long-term, longitudinal measurements of hind limb bone density in the mouse following SCI.

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

A longitudinal examination of bone loss in the femurs and tibiae of adult mice was performed following spinal cord injury using sequential low-dose X-ray scans. Tibia bone loss was detected throughout the study, while bone loss in the femur was not detected until 40 days post injury.

鼠标使用新型脊髓损伤的后肢骨质流失的纵向评价，在体内，方法论

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher ...

An Optimized Procedure for Fluorescence-activated Cell Sorting (FACS) Isolation of Autonomic Neural Progenitors from Visceral Organs of Fetal Mice

During development neural crest (NC)-derived neuronal progenitors migrate away from the neural tube to form autonomic ganglia in visceral organs like the intestine and lower urinary tract. Both during development and in mature tissues these cells are often widely dispersed throughout tissues so that isolation of discrete populations using methods like laser capture micro-dissection is difficult. They can however be directly visualized by expression of fluorescent reporters driven from regulatory regions of neuron-specific genes like Tyrosine hydroxylase (TH). We describe a method optimized for high yields of viable TH+ neuronal progenitors from fetal mouse visceral tissues, including intestine and lower urogenital tract (LUT), based on dissociation and fluorescence-activated cell sorting (FACS). 
The Th gene encodes the rate-limiting enzyme for production of catecholamines. Enteric neuronal progenitors begin to express TH during their migration in the fetal intestine1 and TH is also present in a subset of adult pelvic ganglia neurons2-4 . The first appearance of this lineage and the distribution of these neurons in other aspects of the LUT, and their isolation has not been described. Neuronal progenitors expressing TH can be readily visualized by expression of EGFP in mice carrying the transgene construct Tg(Th-EGFP)DJ76Gsat/Mmnc1. We imaged expression of this transgene in fetal mice to document the distribution of TH+ cells in the developing LUT at 15.5 days post coitus (dpc), designating the morning of plug detection as 0.5 dpc, and observed that a subset of neuronal progenitors in the coalescing pelvic ganglia express EGFP. 
To isolate LUT TH+ neuronal progenitors, we optimized methods that were initially used to purify neural crest stem cells from fetal mouse intestine2-6. Prior efforts to isolate NC-derived populations relied upon digestion with a cocktail of collagenase and trypsin to obtain cell suspensions for flow cytometry. In our hands these methods produced cell suspensions from the LUT with relatively low viability. Given the already low incidence of neuronal progenitors in fetal LUT tissues, we set out to optimize dissociation methods such that cell survival in the final dissociates would be increased. We determined that gentle dissociation in Accumax (Innovative Cell Technologies, Inc), manual filtering, and flow sorting at low pressures allowed us to achieve consistently greater survival (&gt;70% of total cells) with subsequent yields of neuronal progenitors sufficient for downstream analysis. The method we describe can be broadly applied to isolate a variety of neuronal populations from either fetal or adult murine tissues.

An Optimized Procedure for Fluorescence-activated Cell Sorting FACS Isolation of Autonomic Neural Progenitors from Visceral Organs of Fetal Mice

An optimized procedure to purify neural crest-derived neuronal progenitors from fetal mouse tissues is described. This method takes advantage of expression from fluorescent reporter alleles to isolate discrete populations by fluorescence-activated cell sorting (FACS). The technique can be applied to isolate neuronal subpopulations throughout development or from adult tissues.

荧光激活细胞分选（FACS）自主神经祖细胞的分离，从胎鼠的内脏器官的优化程序

During development neural crest (NC)-derived neuronal progenitors migrate away from the neural tube to form autonomic ganglia in visceral organs like the ...

神经科学

Ex Vivo Organoid Model of Adenovirus-Cre Mediated Gene Deletions in Mouse Urothelial Cells

Bladder cancer is an understudied area, particularly in genetically engineered mouse models (GEMMs). Inbred GEMMs with tissue-specific Cre and loxP sites have been the gold standards for conditional or inducible gene targeting. To provide faster and more efficient experimental models, an ex vivo organoid culture system is developed using adenovirus Cre and normal urothelial cells carrying multiple loxP alleles of the tumor suppressors Trp53, Pten, and Rb1. Normal urothelial cells are enzymatically disassociated from four bladders of triple floxed mice (Trp53f/f: Ptenf/f: Rb1f/f). The urothelial cells are transduced ex vivo with adenovirus-Cre driven by a CMV promoter (Ad5CMVCre). The transduced bladder organoids are cultured, propagated, and characterized in vitro and in vivo. PCR is used to confirm gene deletions in Trp53, Pten, and Rb1. Immunofluorescence (IF) staining of organoids demonstrates positive expression of urothelial lineage markers (CK5 and p63). The organoids are injected subcutaneously into host mice for tumor expansion and serial passages. The immunohistochemistry (IHC) of xenografts exhibits positive expression of CK7, CK5, and p63 and negative expression of CK8 and Uroplakin 3. In summary, adenovirus-mediated gene deletion from mouse urothelial cells engineered with loxP sites is an efficient method to rapidly test the tumorigenic potential of defined genetic alterations.

Ex Vivo Organoid Model of Adenovirus-Cre Mediated Gene Deletions in Mouse Urothelial Cells

This protocol describes the process of the generation and characterization of mouse urothelial organoids harboring deletions in genes of interest. The methods include harvesting mouse urothelial cells, ex vivo transduction with adenovirus driving Cre expression with a CMV promoter, and in vitro as well as in vivo characterization.

离体 小鼠尿路上皮细胞腺病毒-Cre介导的基因缺失的类器官模型

Bladder cancer is an understudied area, particularly in genetically engineered mouse models (GEMMs). Inbred GEMMs with tissue-specific Cre and loxP sites ...

癌症研究

Urinary Tract Infection in a Small Animal Model: Transurethral Catheterization of Male and Female Mice

Urinary tract infections (UTI) are extremely common worldwide, incurring significant morbidity and healthcare-associated expenses. Small animal models, which accurately reflect disease establishment and progression, permit dissection of host-pathogen interactions and generation of immunity to infection. In mice, intravesical instillation of uropathogenic E. coli, the causative agent in more than 85% of community acquired UTI, recapitulates many of the stages of infection observed in humans. Until recently, however, UTI could only be modeled in female animals. This limitation has hindered the study of sex-related differences in UTI, as well as other bladder pathologies, such as cancer. Here, we describe a method to instill male mice that allows direct comparison between female and male animals and provide a detailed protocol to assess bladder tissue by flow cytometry as a means to better understand host responses to infection. Together, these approaches will aid in the identification of host factors that contribute to sex biases observed in UTI and other bladder-associated diseases.

The established model of transurethral catheterization of mice allows the study of bladder pathologies, including urinary tract infection, but can only be performed in females. A new model of male transurethral instillation, presented here, will enable research in an area marked by strong clinical and epidemiological differences between the sexes.

小动物模型尿路感染的实验研究: 男性和女性的经尿道插管

Urinary tract infections (UTI) are extremely common worldwide, incurring significant morbidity and healthcare-associated expenses. Small animal models, ...

免疫与感染

A Neurosphere Assay to Evaluate Endogenous Neural Stem Cell Activation in a Mouse Model of Minimal Spinal Cord Injury

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied in vitro using the neurosphere assay. This colony-forming assay is a powerful tool to study the response of NSCs to exogenous factors in a dish; however, this can also be used to study the effect of in vivo manipulations with the proper understanding of the strengths and limitations of the assay. One manipulation of the clinical interest is the effect of injury on endogenous NSC activation. Current models of spinal cord injury provide a challenge to study this as the severity of common contusion, compression, and transection models cause the destruction of the NSC niche at the site of the injury where the stem cells reside. Here, we describe a minimal injury model that causes localized damage at the superficial dorsolateral surface of the lower thoracic level (T7/8) of the adult mouse spinal cord. This injury model spares the central canal at the level of injury and permits analysis of the NSCs that reside at the level of the lesion at various time points following injury. Here, we show how the neurosphere assay can be utilized to study the activation of the two distinct, lineally-related, populations of NSCs that reside in the spinal cord periventricular region - primitive and definitive NSCs (pNSCs and dNSCs, respectively). We demonstrate how to isolate and culture these NSCs from the periventricular region at the level of injury and the white matter injury site. Our post-surgical spinal cord dissections show increased numbers of pNSC and dNSC-derived neurospheres from the periventricular region of injured cords compared to controls, speaking to their activation via injury. Furthermore, following injury, dNSC-derived neurospheres can be isolated from the injury site &#8212; demonstrating the ability of NSCs to migrate from their periventricular niche to sites of injury.

Here, we demonstrate the performance of a minimal spinal cord injury model in an adult mouse that spares the central canal niche housing endogenous neural stem cells (NSCs). We show how the neurosphere assay can be used to quantify activation and migration of definitive and primitive NSCs following injury.

小鼠脊髓损伤模型中内源性神经干细胞活化的干细胞检测

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied ...

Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and peripheral neuroregulation. Although extensive research on the lower urinary tract nervous system has been conducted, most studies have focused on primary culture. This protocol introduces a method for the isolation and culture of bladder neurons and glia from Sprague&#8211;Dawley rats. In this method, the neurons and glia were incubated in a 37 &#176;C, 5% CO2 incubator for 5&#8211;7 days. As a result, they grew into mature shapes suitable for related subsequent immunofluorescence experiments. Cells were morphologically observed using an optical microscope. Neurons, synaptic vesicles, and glia were identified by &#946;-III-tubulin and MAP-2, Synapsin-1, and GFAP staining, respectively. Meanwhile, immunocytochemistry was performed on several neurotransmitter-related proteins, such as choline acetyltransferase, DYNLL2, and SLC17A9.

This protocol attempts to establish a repeatable protocol for primary neurons and glia isolation from rat bladder for further cellular experiments.

来自成人大鼠尿膀胱的原发神经元和胶质的隔离与培养

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and ...

Combined Mechanical and Enzymatic Dissociation of Mouse Brain Hippocampal Tissue

This neural dissociation protocol (an adaptation of the protocol accompanying a commercial adult brain dissociation kit) optimizes tissue processing in preparation for detailed downstream analysis such as flow cytometry or single-cell sequencing. Neural dissociation can be conducted via mechanical dissociation (such as using filters, chopping techniques, or pipette trituration), enzymatic digestion, or a combination thereof. The delicate nature of neuronal cells can complicate efforts to obtain the highly viable, true single-cell suspension with minimal cellular debris that is required for single-cell analysis. The data demonstrate that this combination of automated mechanical dissociation and enzymatic digestion consistently yields a highly viable (&gt;90%) single-cell suspension, overcoming the aforementioned difficulties. While a few of the steps require manual dexterity, these steps lessen sample handling and potential cell loss. This manuscript details each step of the process to equip other laboratories to successfully dissociate small quantities of neural tissue in preparation for downstream analysis.

This neural cell dissociation protocol is intended for samples with a low amount of starting material and yields a highly viable single-cell suspension for downstream analysis, with optional fixation and staining steps.

小鼠脑海马组织机械和酶解离联合

This neural dissociation protocol (an adaptation of the protocol accompanying a commercial adult brain dissociation kit) optimizes tissue processing in ...

从自身免疫小鼠中同时分离所有 CNS 驻留细胞类型

Simultaneous Isolation of Principal Central Nervous System-Resident Cell Types from Adult Autoimmune Encephalomyelitis Mice

Experimental autoimmune encephalomyelitis (EAE) is the most common murine model for multiple sclerosis (MS) and is frequently used to further elucidate the still unknown etiology of MS in order to develop new treatment strategies. The myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) EAE model reproduces a self-limiting monophasic disease course with ascending paralysis within 10 days after immunization. The mice are examined daily using a clinical scoring system. MS is driven by different pathomechanisms with a specific temporal pattern, thus the investigation of the role of central nervous system (CNS)-resident cell types during disease progression is of great interest. The unique feature of this protocol is the simultaneous isolation of all principal CNS-resident cell types (microglia, oligodendrocytes, astrocytes, and neurons) applicable in adult EAE and healthy mice. The dissociation of the brain and the spinal cord from adult mice is followed by magnetic-activated cell sorting (MACS) to isolate microglia, oligodendrocytes, astrocytes, and neurons. Flow cytometry was used to perform quality analyses of the purified single-cell suspensions confirming viability after cell isolation and indicating the purity of each cell type of approximately 90%. In conclusion, this protocol offers a precise and comprehensive way to analyze complex cellular networks in healthy and EAE mice. Moreover, required mice numbers can be substantially reduced as all four cell types are isolated from the same mice.

作者聚焦:通过同时分离所有主要的 CNS 驻留细胞类型来研究神经炎症通路 

To date, protocols for the simultaneous isolation of all principal central nervous system-resident cell types from the same mouse are an unmet demand. The protocol shows a procedure applicable in na&#239;ve and experimental autoimmune encephalomyelitis mice to investigate complex cellular networks during neuroinflammation and simultaneously reduce the required mice numbers.

从成年自身免疫性脑脊髓炎小鼠中同时分离主要中枢神经系统驻留细胞类型

Experimental autoimmune encephalomyelitis (EAE) is the most common murine model for multiple sclerosis (MS) and is frequently used to further elucidate ...

脊髓损伤后小鼠泌尿膀胱分子分析的单细胞分离方法

摘要

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鼠标膀胱壁注射液

鼠标使用新型脊髓损伤的后肢骨质流失的纵向评价，在体内，方法论

荧光激活细胞分选（FACS）自主神经祖细胞的分离，从胎鼠的内脏器官的优化程序

离体小鼠尿路上皮细胞腺病毒-Cre介导的基因缺失的类器官模型

小动物模型尿路感染的实验研究: 男性和女性的经尿道插管

小鼠脊髓损伤模型中内源性神经干细胞活化的干细胞检测

来自成人大鼠尿膀胱的原发神经元和胶质的隔离与培养

小鼠脑海马组织机械和酶解离联合

从成年自身免疫性脑脊髓炎小鼠中同时分离主要中枢神经系统驻留细胞类型

从成年自身免疫性脑脊髓炎小鼠中同时分离主要中枢神经系统驻留细胞类型

脊髓损伤后小鼠泌尿膀胱分子分析的单细胞分离方法

摘要

探索更多视频

鼠标膀胱壁注射液

鼠标使用新型脊髓损伤的后肢骨质流失的纵向评价，在体内，方法论

荧光激活细胞分选（FACS）自主神经祖细胞的分离，从胎鼠的内脏器官的优化程序

离体 小鼠尿路上皮细胞腺病毒-Cre介导的基因缺失的类器官模型

小动物模型尿路感染的实验研究: 男性和女性的经尿道插管

小鼠脊髓损伤模型中内源性神经干细胞活化的干细胞检测

来自成人大鼠尿膀胱的原发神经元和胶质的隔离与培养

小鼠脑海马组织机械和酶解离联合

从成年自身免疫性脑脊髓炎小鼠中同时分离主要中枢神经系统驻留细胞类型

从成年自身免疫性脑脊髓炎小鼠中同时分离主要中枢神经系统驻留细胞类型

离体小鼠尿路上皮细胞腺病毒-Cre介导的基因缺失的类器官模型